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Identification method of plasma membrane protein interactions based on chemical cross-linking mass spectrometry analysis

A technology of chemical cross-linking and identification methods, applied in scientific instruments, analytical materials, material analysis by electromagnetic means, etc. and other problems, to achieve the effect of efficient identification, short time-consuming and efficient processing

Active Publication Date: 2022-08-02
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the above techniques have many limitations: Yeast two-hybrid technique can identify the direct interaction between two proteins, but it is not suitable for complex protein interaction network analysis in vivo, and there is a problem of false positive rate; co-immunoprecipitation technique Although it can identify interacting proteins in vivo, it cannot distinguish between direct and indirect interactions, and it is difficult to achieve effective identification for transient and weak interactions; protein crystallization combined with X-ray diffraction and nuclear magnetic resonance techniques and cryo-electron microscopy techniques can provide High-resolution structural information of protein complexes
However, since some subcellular organelles have similar buoyant densities, the enrichment selectivity of this method for plasma membrane proteins still needs to be improved (Damaraju, S; Zhang, N; Li, N; Tao, L; Damaraju, VL; Dufour, J ; Santos, C; Sun, X J; Mackey, J; Wishart, D S; Cass, C E; Li, L. Anal. Biochem. 2010, 396(1):69), and maintenance of plasma membrane protein interactions cannot be guaranteed

Method used

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  • Identification method of plasma membrane protein interactions based on chemical cross-linking mass spectrometry analysis
  • Identification method of plasma membrane protein interactions based on chemical cross-linking mass spectrometry analysis

Examples

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Effect test

Embodiment 1

[0030] Interaction information identification of HeLa cytoplasmic membrane proteins

[0031] The experimental procedure is as figure 1 shown:

[0032] (1) Acquisition of cells: The HeLa cells in the culture dish were digested with trypsin at 37°C for 2 min, washed three times with 1х phosphate buffer pre-cooled at 4°C, and centrifuged at 3000 rpm for 5 min at 4°C.

[0033] (2) Chemical cross-linking reaction: count the obtained cells, 1х10 7 Cells were added 1mL 1х phosphate buffer (pH8.0) + 1% lysine-specific enrichment impermeable membrane crosslinker (dissolved in dimethyl sulfoxide), the initial concentration of crosslinker was 10mM, organic Phase (dimethyl sulfoxide): water phase = 1:99, room temperature, reaction for 1 h. Centrifuge at 3000rpm, 5min, 4°C, and remove the supernatant.

[0034] (3) Reduction and alkylation: Add the same volume of ammonium bicarbonate with a final concentration of 50 mM to terminate the cross-linking, and at the same time, it contains 50...

Embodiment 2

[0043] Interaction-informative identification of Jurkat cytoplasmic membrane proteins

[0044] (1) Acquisition of cells: The cultured Jurkat cells were washed three times with 1х phosphate buffer solution pre-cooled at 4°C, and centrifuged at 3000 rpm for 5 min at 4°C.

[0045] (2) Chemical cross-linking reaction: count the obtained cells, 1х10 8 Cells were added with 1mL 1х phosphate buffer (pH7.4) + 1% lysine-specific membrane-impermeable cross-linking agent (dissolved in dimethyl sulfoxide), the initial concentration of cross-linking agent was 20 mM, and the organic phase (dimethyl sulfoxide) was added. sulfoxide): water phase volume ratio = 1:99, room temperature, reaction for 1 h. Centrifuge at 3000rpm, 5min, 4°C, and remove the supernatant.

[0046] (3) Reduction and alkylation: Add the same volume of ammonium bicarbonate with a final concentration of 100 mM to terminate the cross-linking, and at the same time, it contains 25 mM tris(2-carboxyethyl) phosphine hydrochlo...

Embodiment 3

[0053] Interaction-informative identification of Escherichia coli plasma membrane proteins

[0054] (1) Acquisition of Escherichia coli: centrifuge 40 mL of Escherichia coli bacteria liquid at 4000 rpm, 4° C., for 6 min. Wash twice with 30mL 1*PBS, centrifuge at 4000rpm, 4°C, 6min.

[0055] (2) Chemical cross-linking reaction: count the obtained Escherichia coli, 1х10 8 Add 1mL 1хphosphate buffer (pH7.8)+1% lysine-specific membrane-impermeable crosslinker (dissolved in dimethyl sulfoxide), the initial concentration of crosslinker is 20mM, the organic phase (dimethyl sulfoxide) sulfoxide): water phase=1:99, room temperature, reaction for 2h. Centrifuge at 3000rpm, 5min, 4°C, and remove the supernatant.

[0056] (3) Reduction and alkylation: Add the same volume of ammonium bicarbonate with a final concentration of 100 mM to terminate the cross-linking, and at the same time, it contains 25 mM tris(2-carboxyethyl) phosphine hydrochloride. Add iodoacetamide with a final concent...

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Abstract

The present invention relates to a method for identifying the interaction of plasma membrane proteins based on chemical cross-linking mass spectrometry analysis. Chemical cross-linking reaction, followed by sample pretreatment operations of reduction, alkylation and enzymatic hydrolysis of plasma membrane proteins, and further mass spectrometry identification and data retrieval of peptide samples, so as to realize plasma membrane protein interaction based on chemical cross-linking mass spectrometry analysis identification. The advantages of the invention are that the experimental operation is simple and fast, the efficient capture of the interaction of the plasma membrane proteins can be realized, and an important technical support is provided for studying the interaction of the plasma membrane proteins.

Description

technical field [0001] The present invention relates to a method for identifying the interaction of plasma membrane proteins based on chemical cross-linking mass spectrometry analysis. The peptide samples were obtained for mass spectrometry identification and data analysis, and the analysis of plasma membrane protein complexes based on chemical cross-linking strategy was realized, which provided important technical support for the study of the spatial structure of plasma membrane proteins and the interaction network of plasma membrane proteins. Background technique [0002] The plasma membrane is a biological membrane that surrounds the outermost layer of cells and consists of lipids and proteins. As the main embodiment of plasma membrane function, plasma membrane proteins have important biological functions such as signal transduction, molecular transport, and cell-cell or cell-matrix information transfer (Wei, X; Song, H; Yin, L; Rizzo, MG; Sidhu, R; Covey, D F; Ory, D S;...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/62G01N33/68
CPCG01N27/62G01N33/6851
Inventor 张丽华赵丽丽赵群李潇杨开广梁振张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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